Phase I of this project was aimed at the development of High Throughput (HT) technology for the discovery of unique, robust, specific enzyme catalysts using expression activity screening. These enzymes are especially valuable for the cost effective synthesis of chiral synthons for use in production of key pharmaceuticals, biological intermediates and natural products as well as for enzyme-based combinatorial drug screening. The HT system developed in Phase I handles up to a million clone assays per week incorporates assay methods for multiple enzyme types and realizes significant discovery rates. Phase I efforts were successful as judged by the discovery of over 100 new enzymes from a diverse group of micro-organisms. A number (27) of the enzymes discovered with the HT system, based upon their novelty, have been taken to commercialization in the form of laboratory scale sampling kits. In Phase II, the discovery effort will continue in an ever-improving HT system as will efforts to commercialize these newly discovered catalysts. Building upon Phase I, Phase II efforts will be directed toward technological improvements in the system to increase assay throughput and to adapt the system to kinetics-oriented assays for directed evolution efforts in enzyme improvement. Rapid methods of clone isolation and indexing for the HT screening effort will be implemented. 96-pin simultaneous pipetting will be incorporated for faster liquid handling and additional fluorescence/absorbance detection systems will be added for higher volume plate reading. Plans also include construction of carousels for plate-feeding to detection systems as part of the effort to incorporate kinetics assays. These additions plus an effort in overall scheduling improvement will enhance the system utility and increase throughput. PROPOSED COMMERCIAL APPLICATION: The proposed research will make available libraries of robust enzyme catalysts to be used for synthesis of optically pure materials. These may include enzymes for synthesis of antibiotics, pharmaceuticals, D and L-natural and non-natural amino acids and medically important natural products. The enzymes will function as selective catalysts under conditions established by industry as optimal for process chemistry.